This is a proposal to develop two novel techniques to eliminate the confounding effects of cellular heterogeneity in the study of psychiatric diseases in the brain. The project will take advantage of the talents of a highly experienced DNA methylome researcher, a creative bioinformatician, and a specialist in triplex forming DNA chemistry. Together, we hope to produce useful bioinformatic and molecular biology tools with immediate applications to existing and future datasets in psychiatric epigenomic research and to ultimately to lay the foundation for a new paradigm of cell type specific disease study. Epigenetic modifications such as DNA methylation are important regulators of genomic function and their misregulation may have etiological significance to psychiatric disease. However, the success of the first round of psychiatric epigenomic studies has been limited with associated disease epigenetic differences displaying seemingly biologically insignificant effects. An intuitive explanation for these findings is that disease relevant epigenetic differences are diluted or skewed by the noise created by cell type specific epigenetic contributions of differing cell types in the brain. To overcome these confounders, in Aim 1 we propose to identify cell type specific epigenetic markers of neurons and glia in order to generate Cell EpigenoType-Specific (CETS) maps enabling the bioinformatic elimination of cell heterogeneity based confounders. We will perform genome wide DNA methylation profiling on the Illumina Infinium 450K microarray platform on post mortem cortex tissue and in FACs separated neurons and glia from MDD (N=20) and control (N=20) samples. CETS markers will be generated using a disease independent unpaired test of neuronal and glial methylation signatures. In Aim 2, the DNA methylation signature of top CETS markers will be validated using pyrosequencing and CETS marker based models will be tested for their ability to normalize varying proportions of mixed neuronal and glial DNA. In the process, we will generate pilot data relevant to the epigenetic contribution to major depressive disorder (MDD) and demonstrate the corrective capability of CETS models in diseased tissue. In Aim 3, we propose to develop a novel tool allowing the sequence and methylation specific purification of DNA that can be used in accordance with CETS marker DNA methylation signals to capture neuronal or glial DNA from a mixed DNA pool. The results of this work will serve as a proof of principle for a more detailed mapping of neuronal cell types over a range of brain regions and may identify epigenetic changes associated with MDD representing prognostic biomarkers or novel targets for epigenetic treatment. CETs model based normalization will greatly reduce the tissue based cellular heterogeneity in psychiatric epigenetic studies, leading to more robust findings. Identified brain cell type specific epigenetic markers in conjunction with TFO based affinity capture will create a powerful tool for the psychiatric epigenetics community to study genomic regulation in small cell populations and to test cell type specific hypotheses using their existing DNA stocks.